Hexavalent chromium (Cr(VI)) or chromate is a major public health concern. Chromates, particularly the insoluble compounds, are well-established human lung carcinogens. The proposed research focuses on the mechanisms of Cr(VI)-induced carcinogenesis, which are currently unknown. Recent studies indicate that particulate Cr(VI) induces chromosome instability, which is often seen in human lung tumors. However, studies of the mechanisms of Cr(VI)-induced chromosome instability have not been done. Our preliminary data show that chronic exposure to particulate Cr(VI) induces centrosome amplification and spindle assembly checkpoint bypass, and thus, the goal of this research is to understand the mechanisms that cause these events leading to Cr(VI)-induced chromosome instability and carcinogenesis. We will test the hypothesis that particulate Cr(VI) induces prolonged G2/M arrest leading to centrosome amplification and spindle assembly checkpoint bypass resulting in chromosome instability and carcinogenesis through four interrelated specific aims: 1) Demonstrate that particulate Cr(VI)-induced DNA double strand breaks cause prolonged G2/M arrest leading to centrosome amplification;2) Identify the molecular mechanisms of how Cr(VI)-induced G2/M arrest uncouples centrosome and cell division causing centrosome amplification focusing on Mps1 and Nek2A;3) Determine the role of Nek2A, Mad1 and Mad2 interactions in particulate Cr(VI)-induced spindle assembly checkpoint bypass;and 4) Identify those cellular and molecular phenotypic changes revealed in Specific Aims 1-3 that occur in cells that escape Cr(VI)-induced death and growth arrest and in cells that are neoplastically transformed by Cr(VI). These four aims will use a combination of established and state-of-the-art toxicological, cytogenetic, and molecular biological techniques with the following approach: 1) Cr(VI)-induced centrosome amplification and spindle assembly checkpoint bypass will be measured with immunofluorescence and cytogenetic assays;2) Gene expression, immunolocalization and RNAi studies will be used to determine the mechanisms of centrosome amplification and checkpoint bypass;and 3) Cell growth and neoplastic transformation assays and studies in human tumors will be used to determine the fate of cells with particulate Cr(VI)-induced chromosome instability. Results will lead to the first reports of detailed information of the interactions of Cr(VI) with centrosome duplication machinery, spindle assembly checkpoint and the first characterizations of chromosome instability in tumors from Cr(VI)-exposed workers. This research is significant because it will provide: 1) An understanding of particulate Cr(VI)'s carcinogenic mechanism;2) Essential information to better assess the risk of exposure to particulates;and 3) A mechanistic approach for further study of Cr(VI), other metals, and lung cancer in general. PUBLIC HEALTH RELEVANCE: Hexavalent chromium (Cr(VI)) is a human lung carcinogen. These studies will advance our basic understanding of the cellular and molecular mechanisms of how cells protect against Cr(VI)-induced genomic instability. Our findings will help us design new treatments and approaches to reduce or prevent Cr(VI)-induced lung cancer and possibly other metals, which continue to be major public health concerns. Finally, we will have established a mechanistic model in a human lung cell system that will allow us to conduct studies for other factors important for preventing human lung cancer both generally and by other agents.